This invention relates to immunoassay methods for the detection or measurement of substances in liquid samples, e.g., biological fluids such as whole blood, serum, plasma, and urine.
A wide variety of substances are commonly detected or measured by immunoassay methods in biological samples; examples are hormones, antibodies, toxins, and drugs. Usually, although not always, either the substance being detected, or a substance used in its detection, is an antibody, hence the term "immunoassay". The antibody is a member of a specific binding pair, the other member of the pair being referred as an antigen or analyte. Other specific binding pairs, besides antibody-antigen pairs, which are measured and used in immunoassays, include pairs of molecules which have specific binding affinity for each other, e.g., hormones--hormone receptors, and biotin-avidin.
Immunoassays are commonly carried out, at least in part, on solid supports, e.g., glass fiber membranes. The two most common formats for immunoassays employing solid supports are competitive and sandwich formats. In a typical competitive assay, the substance to be measured (the analyte) is a low molecular weight substance such as a drug residue or small hormone, with a molecular weight from about 100 to about 2,000; such low molecular weight substances do not easily lend themselves to sandwich assays, described below. In the typical competitive assay, an antibody to the analyte is immobilized on a solid support, and the sample suspected of containing the analyte is brought into contact with that solid support. At the same or a later time, a liquid solution containing labeled analyte is contacted with the support, so that the labeled analyte and any analyte in the sample compete for binding to the immobolized antibody. (If the substance being measured is itself an antibody, the immobilized analyte can be either antibody to that antibody, or an antigen for which that antibody is specific.) The solid support is then washed and the amount of label measured or detected as an inverse measure of analyte in the sample. Typically, the label is a chemiluminescent substance, a radioisotope, or, most preferably, an enzyme which, in the final step, reacts with a chromogenic substrate, which develops color of intensity inversely related to the amount of analyte present in the sample. A typical competitive format is described, e.g., in Litman et al. U.S. Pat. No. 4,540,659, hereby incorporated by reference. In the Litman et al. competitive assay, in addition to the test spot, the solid support also bears what Litman et al. refer to as a "calibration surface" which, preferably, but not necessarily, contains antibody to the substance being measured, and serves as "a standard for the evaluation for the signal level of the measurement surface" (columm 3, lines 14 through 17).
Sandwich immunoassays (e.g., as described in David et al. U.S. Pat. No. 4,376,110, hereby incorporated by reference) generally are used to detect or measure substances (again, analytes) of molecular weights above 2,000, e.g., antibodies and other proteins. In a typical sandwich assay, a first antibody to the analyte is immobilized on a solid support, which is then contacted with the liquid sample so that any analyte in the sample binds to the antibody. A second, labeled antibody to the analyte is then added, the support is washed, and the amount of bound label is measured, bound label being proportional to the amount of analyte in the sample.
A goal of some immunoassays has been a result which is not simply a positive or negative, but which is semiquantitative, i.e., provides a rough, not totally precise estimate of the amount of analyte present in the sample. For example, Chandler et al., International Archives of Allergy and Applied Immunology 72, 267, 1983 describes a semiquantitative immunoassay for IgE in plasma employing three glass capillary tubes, each bearing immobilized antibody specific for IgE. Swanljung European Patent Application No. WO85/02466 describes a colorimetric immunoassay in which the test color is compared to reference colors to provide a semiquantative result. Litman et al., above, states that "one can quantitate the observed results in relation to ratios obtained with known amounts of the analyte and graphing the change in ratio of signal level with change in concentration."